Detección de snps utilizando un nuevo método de ligación acoplada a amplificación isotérmica (lm-rca, ligation mediated-rolling circle amplification)

Resumen

The most common variation in the human genome is a biallelic single nucleotide polymorphism (SNP), which by definition is a DNA sequence change that occurs when a single nucleotide in the genome sequence is altered. For this type of variation to be considered a SNP it must occur in at least 1% of the population. Up to 90% of all human genetic variation take the form of SNPs and are present every 100 to 300 bases along the 3 billion bases of the human genome. LM-RCA is an isothermal and simple Molecular Diagnostic (MD) method that has been optimized as an SNP genotyping tool which allows performing a homogeneous and real time fluorescence detection directly from both whole blood and genomic DNA samples. The LM-RCA reaction is divided into two steps, ligation and subsequent Rolling Circle Amplification (RCA), both at the same temperature. The key of the ligation step is to form a specific circle converting a DNA single stranded linear probe (Open Circle Probe) into a single stranded covalently closed circle by the action of a DNA ligase. This probe will circularize only when hybridized to a DNA region and the DNA ligase will have distinguished between a matched and mismatched single base achieving the SNP discrimination. Upon probe circularization the isothermal exponential RCA reaction begins. This RCA reaction involves an exonuclease (-) DNA polymerase with strand-displacement activity and two primers (reverse and sense) which amplifies exponentially the circular probe. The real time detection of the RCA products is possible by using labeled primers (Amplifluor¿) based on Fluorescence Resonance Energy Transfer (FRET). The development and optimization of this method has enabled the analysis of two SNP mutations, factor V Leiden (G1691A) and prothrombin mutation (G20210A), which alters the genes that codify for the human coagulation factor V and factor II respectively. Despite the fact that different genetic factors that contribute to develop thrombosis have been identified, factor V Leiden and prothrombin mutation show a higher prevalence among them, thus the importance to develop suitable and alternative methods to achieve an accurate genotyping analysis.